Limb Girdle Kas Distrofisi 2R (LGMD2R)'de Mekanotransdüksiyonun Rolünün Araştırılması
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Ultra-rare LGMD2R (MIM 601419) is a late onset disease with slow progression caused by a splice site mutation on desmin protein leading to interruption of laminB binding domain. Mechanotransduction is a process which cells use to sense and convert mechanical stimuli into biochemical signals. Defective mechanotransduction signal effects several disease pathogenesis including muscular dystrophies. To determine impaired mechanotransduction signaling due to loss of desmin-laminB interaction, mechanosensitive pathways and genes were investigated. In order to study the effect of loss of mechanosensitivity, control and patient cells were subjected to cyclic stretch (mechanical load) by using Flexcell Tension system. Firstly, MAPK pathway was investigated and no significant change was observed both in ERK1/2 and its downstream genes. Only EGR1 expression which is a mechanosensitive gene was found to be downregulated in LGMD2R. Secondly, YAP as key regulator of skeletal muscle mechanotransduction was investigated. Since YAP translocates to nucleus under stress conditions, it was found that 37.5% of myonuclei were YAP positive in control skeletal muscle tissue while in patient it is reduced to 15.1%. Furthermore, in resting state, patient’s myotubes have 1,5 times higher p-YAP which is the inactive form. After application of cyclic stretch, the amount of p-YAP and t-YAP were declined simultaneously in the patient. Moreover, the expression levels of some YAP target genes were analyzed. They all responded to mechanical stretch by means of upregulation in the patient. However, when control and patient compared, CTGF and C-MYC expression was downregulated while DIAPH1 and MYL9 were not changed. Taken as a whole, the data suggested reduced basal YAP activity in the patient, reduced ability of LGMD2R cells to challenge mechanical load although patient cells were still able to respond to a mechanical load. Interestingly, ANKRD1 and S isoform of ANKRD2 were upregulated in the patient. Hence, elevation of ANKRD1 and S isoform of ANKRD2 may also reflects defense against loss of mechanosensitivity in LGMD2R. This thesis was funded by TÜBİTAK (214S174).